Polarized vesicles

Epithelial cells are interconnected at the apical (mucosal) side by a complex network of proteins, called the tight junctions (TJ). First thought to have merely a static role in restricting access of compounds present in the luminal fluid to the underlying subepithelial tissue and systemic circulation by the paracellular pathway, TJ are known today to be dynamic structures involved in cellular differentiation, cell signaling (Harder and Margolis 2008), polarized vesicle trafficking, and protein synthesis. 

Supramolecular chemists without BLM-workstation can determine gating charges in polarized vesicles (Fig. 11.8), using the HPTS assay for synthetic ion channels and the ANTS/DPX assay for synthetic pores (Fig. 11.5) [7]. To polarize vesicles, an inside-negative Nernst potential is applied with a potassium gradient (Eq. 11.6), osmotically balanced with sodium, coupled with the potassium carrier valinomycin at intermediate concentrations sufficient for rapid potential buildup without immediate collapse, and monitored by an emission increase of the externally added probe safranin O (Fig. 11.8a and b). 

Planar bilayer condnctance experiments and fluorescence kinetics in polarized vesicles provide access to I—V curves and gating charges. In single-channel conductance experiments, Zg is classically determined from changes in the open probability Pg with applied voltage, but correct... 

Polarized vesicles are convenient and reliable systems to record I-V curves and gating charges. (Figure 12b). 

Figure 17 Techniques to study (a) catal3dic pores in unpolarized or polarized vesicles with entrapped fluorogenic substrates and (b) covalent modifications of ion channels and pores in single-channel conductance expaiments.

Phospholipids are a major component of all biological membranes together with glycolipids and cholesterol. Due to their polar nature, i.e. hydrophilic head and hydrophobic tail, phospholipids form in water vesicles or liposomes. 

Due to their physicochemical properties trace amines can pass the cell membrane to a limited extent by passive diffusion, with the more lipophilic PEA and TRP crossing membranes more readily than the more polar amines TYR. and OCT. In spite of these features, trace amines show a heterogeneous tissue distribution in the vertebrate brain, and for TYR. and OCT storage in synaptic vesicles as well as activity-dependent release have been demonstrated. So far, trace amines have always been found co-localized with monoamine neurotransmitters, and there is no evidence for neurons or synapses exclusively containing trace amines. 

Just as myosins are able to move along microfilaments, there are motor proteins that move along microtubules. Microtubules, like microfilaments, are polar polymeric assemblies, but unlike actin-myosin interactions, microtubule-based motors exist that move along microtubules in either direction. A constant traffic of vesicles and organelles is visible in cultured cells especially using time-lapse photography. The larger part of this movement takes place on micrombules and is stimulated by phorbol ester (an activator of protein kinase C), and over-expression of N-J aj oncoprotein (Alexandrova et al., 1993). 

We have proposed that vesicle aggregation is probably related to the disposition of pardaxin bound in the phosphatidylserine vesicle lipid bilayer (26). This conclusion is supported by the observation that phosphatidycholine vesicles are not induced to aggregate and that the pardaxin-induced phosphatidylserine vesicle aggregation is affected by charge polarization of the vesicle (26). This suggestion seems to be consistent also with the voltage dependence of fast "pore" activity of pardaxin, the channels which are open only at positive membrane potentials. 

Transferosomes represent another system of encapsulation using ultradeformable vesicle carriers for bioactive molecules, applied until now for direct transdermal drug delivery. They are built from polar lipids and have high flexibility, and are rich in unsaturated fatty acids and carotenoid pigments." ... 

The availability of the purified transporter in large quantity has enabled investigation of its secondary structure by biophysical techniques. Comparison of the circular dichroism (CD) spectrum of the transporter in lipid vesicles with the CD spectra of water-soluble proteins of known structure indicated the presence of approximately 82% a-helix, 10% ) -turns and 8% other random coil structure [97]. No / -sheet structure was detected either in this study or in a study of the protein by the same group using polarized Fourier transform infrared (FTIR) spectroscopy [98]. In our laboratory FTIR spectroscopy of the transporter has similarly revealed that... 

Unilamellar vesicles (PC, aT) Polar head/acyl core Chemical reaction, aT-DPPH 26 446... 

Unilamellar vesicles PC Polar head/acyl core Fluorescence polarization (DSHA) 33 381... 

Unilamellar vesicles, PC Hydrocarbon core Fluorescence polarization (AS) 2 381... 

Solute uptake can also be evaluated in isolated cell suspensions, cell mono-layers, and enterocyte membrane vesicles. In these preparations, uptake is normalized by enzyme activity and/or protein concentration. While the isolation of cells in suspension preparations is an experimentally easy procedure, disruption of cell monolayers causes dedifferentiation and mucosal-to-serosal polarity is lost. While cell monolayers from culture have become a popular drug absorption screening tool, differences in drug metabolism and carrier-mediated absorption [70], export, and paracellular transport may be cell-type- and condition-depen-dent. 

---